Elsevier

Toxicology

Volume 337, 4 November 2015, Pages 39-46
Toxicology

Iron overload triggers mitochondrial fragmentation via calcineurin-sensitive signals in HT-22 hippocampal neuron cells

https://doi.org/10.1016/j.tox.2015.08.009Get rights and content

Highlights

  • FAC-induced iron overload promotes neuronal apoptosis.

  • Iron overload causes mitochondrial fragmentation in a Drp1-dependent manner.

  • Iron-induced Drp1 activation depends on dephosphorylation of Drp1(Ser637).

  • Calcineurin is a key regulator of Drp1-dependent mitochondrial fission by iron.

Abstract

The accumulation of iron in neurons has been proposed to contribute to the pathology of numerous neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. However, insufficient research has been conducted on the precise mechanism underlying iron toxicity in neurons. In this study, we investigated mitochondrial dynamics in hippocampal HT-22 neurons exposed to ferric ammonium citrate (FAC) as a model of iron overload and neurodegeneration. Incubation with 150 μM FAC for 48 h resulted in decreased cell viability and apoptotic death in HT-22 cells. The FAC-induced iron overload triggered mitochondrial fragmentation, which was accompanied by Drp1(Ser637) dephosphorylation. Iron chelation with deferoxamine prevented the FAC-induced mitochondrial fragmentation and apoptotic cell death by inhibiting Drp1(Ser637) dephosphorylation. In addition, a S637D mutation of Drp1, which resulted in a phosphorylation-mimetic form of Drp1 at Ser637, protected against the FAC-induced mitochondrial fragmentation and neuronal apoptosis. FK506 and cyclosporine A, inhibitors of calcineurin activation, determined that calcineurin was associated with the iron-induced changes in mitochondrial morphology and the phosphorylation levels of Drp1. These results indicate that the FAC-induced dephosphorylation of Drp1-dependent mitochondrial fragmentation was rescued by the inhibition of calcineurin activation. Therefore, these findings suggest that calcineurin-mediated phosphorylation of Drp1(Ser637) acts as a key regulator of neuronal cell loss by modulating mitochondrial dynamics in iron-induced toxicity. These results may contribute to the development of novel therapies for treatment of neurodegenerative disorders related to iron toxicity.

Introduction

Iron, which is an essential cofactor for almost all living organisms, is involved in a wide range of cellular processes, including DNA synthesis and repair, energy metabolism, phospholipid metabolism, and oxidative phosphorylation (Salvador 2010). In addition, iron is crucial for neuronal development and neurotransmitter production in the brain (Sipe et al., 2002). However, the accumulation of iron impairs brain function, and the dysregulation of iron metabolism results in tissue damage due to the formation of free radicals, such as the hydroxyl radical (radical dotOH), which is a reactive oxygen species (ROS) intermediate that is highly toxic in most biological systems (Lipinski, 2011, Urrutia et al., 2014). Several studies have indicated that increased iron levels are associated with neurodegenerative physiology, including those underlying Alzheimer’s disease and Parkinson’s disease (Gotz et al., 2004, Rogers et al., 2002). Therefore, mounting evidence suggests that the accumulation of iron in particular areas of the brain is a hallmark of neurodegenerative disease (Zecca et al., 2004). However, the precise toxicological mechanisms of iron overload are not fully understood.

Mitochondria are highly dynamic organelles that continuously fuse and divide through the processes of fusion and fission, respectively. Increases in the fusion events produce elongated mitochondria, whereas fission results in fragmented mitochondria. A balance between mitochondrial fusion and fission is important in cellular function (Westermann 2010). Mitochondrial dynamics are involved in numerous cellular processes, including the homeostasis of mitochondrial biogenesis, mitochondrial turnover, mitochondrial distribution, cell division, and cell death (Westermann 2010). In mammalian cells, mitochondrial fusion is regulated by mitofusin 1 and 2 (Mfn1/2) and optic atrophy protein 1 (Opa1), whereas fission is controlled by dynamin-related protein 1 (Drp1) and mitochondrial fission 1 (Fis1) (Knott et al., 2008, Westermann, 2010). An increasing number of reports have proposed that abnormalities in the mitochondrial dynamics of neuronal cells are involved in various neurodegenerative pathological processes (Cho et al., 2010). Although mitochondria are important organelles in all cell types, they are particularly important in the brain because neuronal cells are highly dependent on mitochondrial energy production (Du et al., 2012). In addition, deficits in neuronal energy supply have been linked to neuronal cell death, and changes in mitochondrial dynamics significantly influence almost all aspects of mitochondrial function, including energy metabolism (Knott et al., 2008, Westermann, 2010). Accordingly, mitochondrial dynamics are considered an important target of protection in neurodegenerative diseases. Several lines of investigation have reported an association between mitochondrial function and iron accumulation in various neurodegenerative diseases. Because mitochondrial function is crucially dependent on adequate iron supply (Connor et al., 2001, Sipe et al., 2002), a disruption of iron homeostasis causes damage to mitochondrial DNA (mtDNA) and a loss of respiratory capacity (Gao et al., 2009, Gille and Reichmann, 2011). However, the precise mechanistic pathways underlying the association between iron dysregulation and mitochondrial dynamics are not fully understood.

Previous reports have shown that calcium (Ca2+) signals affect mitochondrial function by governing mitochondrial dynamics through the activation of Ca2+-sensitive downstream effectors, such as Ca2+/calmodulin-dependent protein kinase 1 (CaMKI) and the protein phosphatase calcineurin (Cereghetti et al., 2008, Han et al., 2008, Munoz et al., 2011). Calcineurin, which is a Ca2+- and calmodulin-dependent serine/threonine phosphatase that belongs to the protein phosphatase 2B family (Klee et al., 1979), is considered an important mediator of cellular signaling, and it is involved in processes, such as T-cell activation, cell death, and the dephosphorylation of target proteins, including transcription factors (de la Pompa et al., 1998, Liu et al., 1991, Yazdanbakhsh et al., 1995). The activation of calcineurin is involved in mitochondrial fission through the dephosphorylation of Drp1, which is the activated form of Drp1 (Cereghetti et al., 2008). Moreover, the inhibition of calcineurin activity prevents the activation of Drp1-dependent mitochondrial fission and cell death (Pennanen et al., 2014, Slupe et al., 2013). Recently, iron overload has been shown to affect the calcineurin-dependent regulation of the nuclear factor of activated T-cells signaling pathway (Lin et al., 2013). However, the precise mechanisms underlying how iron accumulation is involved in the regulation of mitochondrial morphology through calcineurin signaling in neuronal cells are still unclear.

In this study, we examined the relationship between iron-induced toxicity and mitochondrial dynamics in neuronal cells. HT-22 hippocampal neuron cells were exposed to different concentrations of ferric ammonium citrate (FAC). FAC caused cellular apoptosis accompanied by mitochondrial fragmentation induced in a Drp1-dependent manner. In addition, we found that the activation of Drp1 was regulated by a calcineurin-sensitive signal.

Section snippets

Materials

FAC, deferoxamine (DFO), cyclosporin A (CsA), and Tacrolimus (FK506) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture and treatment

HT-22 cells, derived from HT-4 cells that were immortalized from primary mouse hippocampal neuronal cultures (Davis and Maher 1994), were maintained in Dulbecco’s modified Eagle’s medium (Welgene, Daegu, Korea) that was supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Welgene) at 37 °C in a humidified 5% CO2 incubator

Statistical analysis

The data are presented as the mean and standard deviation of the results from three independent experiments (n = 3). The statistical significance of the experimental differences was determined with two-way analysis of variance with GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). P values less than 0.05 were considered statistically significant, and significance is indicated on the graphs with an asterisk. P values less than 0.01 and 0.001 are indicated with two and three

FAC-induced iron overload promoted neuronal apoptosis

In order to confirm the neuronal toxicity of iron, we used FAC as a source of iron. We first assessed the changes in iron concentration following the dose- (75–300 μM) and time- (24–72 h) dependent exposure of HT-22 cells to FAC. The concentration of iron increased significantly over the 150 μM of FAC exposure for 48 h. Moreover, the concentration of iron was consistently increased from 24 h of FAC exposure (Supplementary Fig. 1). In addition, we assessed the effects of FAC on the cell viability of

Discussion

Iron toxicity in the brain has been implicated in the pathogenesis of neurodegenerative disorders, and iron chelation has been demonstrated to reduce the progression of the neurological symptoms of these disorders (Andersen et al., 2014, Zecca et al., 2004). Although iron is an essential cofactor in the oxidative metabolism of mitochondria, excess iron has been implicated in mitochondrial dysfunction and loss of neuronal cells (Beal, 1998, Connor et al., 2001, Rouault, 2012). Moreover, abnormal

Acknowledgments

This research was supported by grants (NRF-2014R1A2A1A11054095 and NRF-2015R1A4A1042271) from the National Research Foundation of Korea, a grant from the government of the Republic of Korea, and a grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Researchinitiative program (KGM4611512), Republic of Korea.

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      Citation Excerpt :

      Second, in neuron, iron overload-induced mitochondrial fission is probably linked with calcium signaling, as DRP1 activity has been shown to be regulated by cellular calcium levels. For example, elevated iron levels appear to accelerate calcineurin activity along with increased intracellular calcium levels, which could upregulate mitochondrial fission by DRP1 dephosphorylation at serine 637 and affect neurodegeneration (Park et al., 2015; Lee et al., 2016). Collectively, altered mitochondrial dynamics are hallmarks of neurodegenerative diseases that are affected by either iron deficiency or overload, although more studies are warranted to fully understand a causal relationship between iron status and mitochondrial fission-fusion balance in neuronal cells.

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    These authors contributed equally to the study.

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